Molecular cloning and characterization of two 1-deoxy-D-xylulose-5-phosphate synthase genes involved in tanshinone biosynthesis in Salvia miltiorrhiza

The synthesis of diterpenoid tanshinones, which are some of the main bioactive ingredients of Danshen (the roots of Salvia miltiorrhiza), begins with the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) catalyzes the first step of the MEP pathway and is considered to be a key rate-limiting enzyme. To confirm the function of DXS genes, in this study two isoforms of DXS (SmDXS1 and SmDXS2) were isolated by rapid amplification of DNA ends from leaves of S. miltiorrhiza. Bioinformatic analyzes indicated that two SmDXSs had high homology with other plant DXSs. Tissue expression pattern analysis revealed that SmDXS1 and SmDXS2 exhibited distinct expression patterns in S. miltiorrhiza. SmDXS1 was expressed in all analyzed tissues; the highest expression was observed in leaves, followed by stems, with weak expression in roots. In contrast, SmDXS2 transcripts were predominantly detected in roots, but very weakly. Subcellular localization analysis revealed that SmDXS1 and SmDXS2 were both localized in the chloroplast. Agrobacterium-mediated transformation was used for induction of transgenic hairy root lines and tanshinone yield was assessed by high-performance liquid chromatography. Compared with the control, overexpression of SmDXS1 and SmDXS2 significantly enhanced tanshinone accumulation in transgenic roots, whereas only down-regulation of SmDXS2 led to a significant decrease. Our results indicated that SmDXS2 was an important rate-limiting gene in the MEP pathway involved in tanshinone biosynthesis and could be an effective regulatory target for controlling tanshinone accumulation in S. miltiorrhiza hairy root cultures.